Marigold male inbred line denominated KI4662

ABSTRACT

A new and distinct inbred plant of  Tagentes erecta  named KI4662 and characterized by elevated levels of zeaxanthin and little or no lutein.

RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Ser. No.62/117,164 filed Feb. 17, 2015.

FIELD OF THE INVENTION

The present invention relates generally to marigolds (Tagetes erecta L.)and, more specifically, to a novel marigold male parent inbred linedenominated KI4662 which has high levels of zeaxanthin and little or nolutein.

BACKGROUND OF THE INVENTION

Xanthophylls are yellow pigments that form one of two major divisions ofthe carotenoid group. Their molecular structure is similar to carotenes,which form the other major carotenoid group division, but xanthophyllscontain oxygen atoms, while carotenes are purely hydrocarbons with nooxygen. Xanthophylls contain their oxygen either as hydroxyl groupsand/or as pairs of hydrogen atoms that are substituted by oxygen atomsacting as a bridge (epoxide).

Like other carotenoids, xanthophylls are found in highest quantity inthe leaves and/or flowers of most green plants, where they act tomodulate light energy. The xanthophylls found in the bodies of animals,and in dietary animal products, are ultimately derived from plantsources in the diet. For example, the yellow color of chicken egg yolks,fat, and skin comes from ingested xanthophylls (primarily lutein, whichis often added to chicken feed for this purpose).

The yellow color of the human macula lutea in the retina of the eyecomes from the lutein and zeaxanthin it contains, both xanthophyllsagain requiring an exogenous source in the human diet to be present inthe eye. These function in eye protection from ionizing blue light,which they absorb.

The group of xanthophylls includes (among many other compounds) lutein,zeaxanthin, neoxanthin, violaxanthin, and α- and β-cryptoxanthin.

There is an interest in developing products that have high levels ofxanthophylls. For example, xanthophylls are known to have antioxidantproperties and have been shown to prevent age-related maculardegeneration (AMD).

Marigold flowers are the most commercially significant source ofxanthophylls. A prominent supplier of xanthophyll pigments extracts themfrom a commercial marigold hybrid (Hybrid 50011, U.S. Pat. No.6,894,208) which typically has a total xanthophyll composition of 94%lutein (L) and 6% zeaxanthin (Z); with an average L:Z ratio range of15:1. Marigold hybrids that produce zeaxanthin as the primaryxanthophyll, with minimal lutein, would provide an agronomicallyfeasible source of zeaxanthin for the human dietary supplement industry.Chrysantis, Inc. (West Chicago, Ill.) has developed a high zeaxanthinmarigold hybrid via mutagenesis.

SUMMARY OF THE INVENTION

The invention consists of a marigold male parent inbred line withaltered carotenoid compositions through a chemically-induced mutationbreeding process. The mutagenesis program identified three commerciallyuseful mutant plants including a high zeaxanthin mutant plantKI-TeM₂-4662 among several altered carotenoid mutants during 2010-2012.Mutant plant KI-TeM₂₀₄₆₆₂ was selfed to develop a stable mutant inbredline KI4662. Development of a proprietary, high flower yielding highzeaxanthin marigold hybrid from KI4662 with the potential toaccumulate >3 mg/g of zeaxanthin and little to no lutein is highlyvaluable in developing an improved higher yielding marigold source ofzeaxanthin.

The mutant KI-TeM₂₀₄₆₆₂ was selfed to fix the phenotype andsimultaneously crossed with a female line to produce seed of aheterozygous mutant hybrid. Stabilization of the phenotype inKI-TeM₂₀₄₆₆₂ was achieved via selfing selected individuals (with >3 mg/gof zeaxanthin) for six generations. Inbreeding via selfing alsoconsiderably reduced the frequency of the undesirable weak phenotypes inthe later generations. All the individuals within the selfed generationsshowed the mutant chemotype with an average zeaxanthin content of 3.4±4mg/g. KI4662 represents a unique assemblage of homozygous allelesthroughout the genome as a consequence of single seed descent selfing toinbred status. Genetic analysis revealed that the mutation for highzeaxanthin in KI4662 was recessive and did not express fully inheterozygous hybrid combinations. The wild type originating germplasm,Scarletade (hereinafter referred to as “SCR”) also showed a partialdominance for carotenoid expression in a hybrid combination. Thedevelopment of a high zeaxanthin marigold hybrid will require the mutantallele to be present in both the male and female parent lines. Amolecular marker-assisted introgression program results in the transferof the mutant allele from the male parent into the female parent.

Plants of the inbred line KI4662 have not been observed under allpossible environmental conditions. The phenotype may vary somewhat withvariations in environment and culture such as temperature, lightintensity, day length, water status, and/or fertilizer rate or typewithout, however, any variance in genotype.

An object of the present invention is a marigold plant with a high levelof zeaxanthin and little or no lutein for use as an antioxidant in humanand animal food, beverages and personal care products.

Another object of the invention is an inbred line of Tagetes erecta thatis novel, stable, and uniform and has good agronomic characteristicsthat confers the trait of hyper-accumulation of zeaxanthin and little orno lutein to its progeny.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chromatogram of SCR (left) and mutant KI4662 (right).

FIG. 2 is a scheme of the development of advanced generations of mutantplant KI4662 leading to inbred line KI4662.

DESCRIPTION OF THE INVENTION

The term “M” generation as used herein is the seed (and resultingplants) exposed to a mutagenic agent, “M₂” is the progeny ofself-pollinated M₁ plants, “M₃” is the progeny of a selected singleself-pollinated M₂ plant, and so on. The term “progeny” refers to theplants and seeds of all subsequent generations resulting from a selfedplant from the previous generation.

African marigold (Tagetes erecta L.) flowers are commercially extractedand utilized as a natural source of xanthophyll pigments for the poultryand human dietary supplement industries. A prominent supplier ofxanthophyll pigments extracts them from a commercial marigold hybrid(Hybrid 50011, U.S. Pat. No. 6,894,208) which typically has a totalxanthophyll composition of 94% lutein (L) and 6% zeaxanthin (Z); with anaverage L:Z ratio range of 15:1. Marigold hybrids that producezeaxanthin as the primary xanthophyll, with minimal lutein, wouldprovide an agronomically feasible source of zeaxanthin for the humandietary supplement industry. Chrysantis, Inc. (West Chicago, Ill.) hasdeveloped a high zeaxanthin marigold hybrid via mutagenesis. The hybridrequires the mutant allele to be present in both parents. Carefulintrogression of the mutant allele of the present invention into bothparental lines using marker-assisted introgession and backcrossing willfacilitate the development of a higher flower yielding high zeaxanthinmarigold hybrid.

In pursuit of the present invention, a large-scale chemical mutagenesisprogram was conducted using ethyl methane sulfonate (EMS) as a mutagenicagent in a publicly available elite marigold open pollinated varietybackground, Scarletade. More than 4000 first generation (M₁) mutantplants were generated during the first two years of the program.Subsequently about 9000 second generation mutants (M₂) derived from 1500M₁ families were screened for their L:Z ratios and their carotenoidprofile during the program. An optimized, rapid protocol called the ‘NoYield Incomplete Saponification’ (NYIS) method was used for rapidscreening of these 9000 M₂ plants for their L:Z ratios. Each M₂ seed wasgrown to produce M₂ flowering plants. Flowers from each M₂ plant werecollected, freeze dried and quantitated for carotenoid levels using HPLCbased, industry standard AOAC method. Briefly, 0.5-1 g ground petalsamples were extracted and saponified with 30 mL HEAT extraction solvent(hexane, ethanol, acetone, and toluene in volume ratios of 10:6:7:7) and2 mL 40% methanolic potassium hydroxide (w/v) in a 56° C. water bath setto shake at 120 rpm for 20 minutes. The extract was analyzed in an HPLCwith a Waters Sphersorb CN column (50×4.6 mm, 3 μm). An isocraticelution with mobile phase of hexanes (74.6%), methylene chloride(24.9%), methanol (0.4%), n,n-diisopropylethylamine (0.1%) at 1.000mL/min flow rate was used. The column temperature was maintained at 25°C. and 10 μL of sample was injected to run for 13 minutes per assay. Thediode array detector (DAD) was set at 446 nm. A standard method was usedfor the quantitation of lutein and zeaxanthin.

The large scale screening of M₂ plants identified 25 mutants withaltered carotenoid profile of which five mutants (KI132, KI935, KI4656,KI4662 and KI4871) had potentially valuable alterations to their L:Zratio. KI132 is a mutant with a 6:1 L:Z ratio and is close to apotentially valuable AREDS2 target of 5:1. KI935, KI4656 and KI4662 arezeaxanthin enriched (high zeaxanthin) mutants, with almost no lutein tovery low amounts of lutein present in the carotenoid profile. MutantsKI935 and KI4656 were not advanced further due to flower sterilityissues. KI4662 was advanced from M₂ to M₇ stage using a single-seeddescent procedure as shown in FIG. 2. Briefly KI4662 identified as asingle M₂ plant, was naturally self-pollinated in isolation to produceM₃ seed. Eighty M₃ plants were generated from M₃ seed and flowers werequantitated for lutein and zeaxanthin content using the AOAC method.Based on the carotenoid profile, 2-3 M₃ plants with the highestzeaxanthin content were selected and naturally self-pollinated inisolation to produce M₄ seed. Using the same procedure, KI4662 wasadvanced from the M₂ to the M₆ generation producing M₇ seed.

The benefits of lutein, especially for eye health have been well studiedfor decades. Research shows that zeaxanthin along with lutein also playsa unique, distinctive role in maintaining optimal eye health. Inaddition, human studies have demonstrated that both lutein andzeaxanthin are present in the skin. Animal studies have providedevidence of lutein and zeaxanthin protecting skin from light-inducedskin damage, especially the ultraviolet wavelengths. The unique role ofzeaxanthin in addition to lutein in human dietary requirements has beenextensively studied. In this context, the high zeaxanthin mutant linessuch as KI4662 with a potential to accumulate up to 3.4±0.2 mg/g ofzeaxanthin with little to no lutein would be highly valuable.

The carotenoid composition of a typical marigold flower contains >85%lutein and 5% zeaxanthin. The current commercially dominant luteinmarigold hybrid on average yields more lutein (18 mg/g) than zeaxanthin(1.5 mg/g) on a dry matter basis. Some high zeaxanthin hybrids have beendeveloped but they contain measurable amounts of lutein. In thiscontext, developing high zeaxanthin hybrids utilizing mutant inbred lineKI4662 as a male parental line would add great economic value indeveloping zeaxanthin based products as the resulting hybrid would havehigher overall flower yield, equivalent zeaxanthin on a dry matter basisand almost no lutein. Growing mutant variety KI4662 in a hybrid formwill significantly increase flower yield relative to the per se inbredwhile also having the altered carotenoid profile change relative to thewild type parent.

The average lutein and zeaxanthin content in flowers of SCR isapproximately 22.2±2.3 mg/g and 1.66±0.3 mg/g respectively undergreenhouse conditions. Zeaxanthin content in mutant KI4662 is almostdouble, compared to wild type SCR, under the greenhouse conditions. Incontrast, the lutein content in mutant line KI4662 is negligible (<0.05mg/g) compared to the 22.2 mg/g present in the wild type SCR (FIG. 1).Ball Horticultural Company has developed high zeaxanthin mutants and hasdemonstrated that the high zeaxanthin mutation is on the lut2 gene whichcodes for lycopene ε-cyclase (personal discussion) causing constitutive,whole plant lutein deficiencies. The mutation of mutant KI4662 is likelyto be a lut2 mutation (lutein-deficient mutant) which is characterizedby very low lutein levels in the leaves as well as in flowers throughoutthe entire plant. Studies have shown that the lut2 mutation eliminateslutein production and is biochemically and genetically consistent with adisruption in the gene encoding the enzyme lycopene ε-cyclase (Pogson BJ, McDonal K A, Truong M, Britton G and DellaPenna D. 1996. Arabidopsiscarotenoid mutants demonstrate that lutein is not essential forphotosynthesis in higher plants. The Plant Cell, Vol. 8, 1627-1639;Pogson B J, Niyogi K K, Bjorkman O, DellaPenna D. 1998. Alteredxanthophyll compositions adversely affect chlorophyll accumulation andnonphotochemical quenching in Arabidopsis mutants. PNAS. 95;13324-13329).

In accordance with one aspect of the present invention, provided is aninbred marigold seed and plants thereof designated KI4662. The presentinvention further relates to a method for producing inbred marigoldseeds that includes, but is not limited to, the steps of planting seedof inbred marigold KI4662 in proximity to itself or to different seedfrom a same family or line, growing the resulting marigold plants,cross-pollinating the resulting marigold plants, and harvestingresultant seed obtained from such inbred plants using techniquesstandard in the agricultural arts such as would be necessary to bulk-upseed such as for hybrid production. The present invention also relatesto inbred seed produced by such a method.

In any cross between inbred marigold plant KI4662 and another inbredmarigold plant, KI4662 may be designated as the male (pollen parent) orthe female (seed parent). The present invention also relates to amarigold plant that expresses substantially all of the physiological andmorphological characteristics of inbred marigold plant KI4662 and to asubstantially homogenous population of marigold plants having all thephysiological and morphological characteristics of inbred marigold plantKI4662. Any marigold plants produced from inbred marigold plant KI4662are contemplated by the present invention and are, therefore, within thescope of this invention. A description of physiological andmorphological characteristics of marigold plant KI4662 is presented inTable 2.

It should be appreciated by one having ordinary skill in the art that,for the quantitative characteristics identified in Table 2, the valuespresented are typical values. These values may vary due to theenvironment and accordingly, other values that are substantiallyequivalent are also within the scope of the invention.

Inbred marigold line KI4662 shows uniformity and stability within thelimits of environmental influence for the traits described in Table 2.Inbred KI4662 has been self-pollinated a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure the homozygosity and phenotypic stability necessary to use inlarge scale, commercial production. The line has been increased both byhand in isolated fields with continued observations for uniformity. Novariant traits have been observed or are expected in KI4662

The plants of the present invention have the taxonomic description ofbeing genus Tagetes, species erecta, family Asteraceae and the commonname marigold. Table 2 sets out the characteristic traits of KI4662 andTable 3 sets out the definitions of the traits of Table 2.

TABLE 2 Characteristics of KI4662 at vegetative and flowering stagePloidy Diploid Chromosome number 24 Greenhouse Observations FieldObservations Average Range Average Range Growth Form Upright Plantheight at maturity 34.9 cm 31-38 cm 56.5 cm 50-67 cm (Vegetative) (56days) Plant height class (flowering) Tall Tall Flowering SeasonMid-Season Mid-Season Flower Type Chrysanthemum Chrysanthemum FlowerFullness Semi-double Semi-double Silhouette Flattened Flattened Numberof Flower heads per 8.5 13.6 8-25 plant (132 days) Flower Head Diameter6.5 cm 5.6 cm 4.1-6.2 cm Flower Odor Mild Mild Mild “Marigold”“Marigold” “Marigold” Flower Head Color Orange Orange Orange Days fromplanting to first 95 days 90-100 days 85 days 80-90 days flower Lengthof flowering season 140 days 135-145 days 120 days 115-132 days Plantheight at maturity 110.1 cm 102-118 cm 98.5 cm 92-108 cm (Flowering) (98days) Plant Width at maturity 235.3 cm² 161.2-272.6 cm² (Vegetative) (56days) Plant width class (flowering) Semi-compact Semi-compact Leaf TypeCompound Compound Leaf Shape lanceolate lanceolate Leaf Margin DentateDentate Leaf Width 4.9 cm 3.0-6.0 cm Leaf Length 15.6 cm 13.0-18.0 cmLeaf Color Medium green Medium green Petiole Anthocyanin Mild Mild StemProfile Straight Straight Stem Structure Intermediate Intermediate NodeLength (middle of 4.2 cm 3-5 cm 5.3 cm 4.5-7 cm plant) (vegetative) (56days) Node Length (middle of 4.8 cm 3-6 cm 5.26 cm 4.58-5.55 cm plant)(flowering)(98 days) Number of internodes below 1 1 first branch Numberof first order 8.9  6-10 8 6-10 branches (vegetative)(56 Days) Number offirst order 36.3 32-50 8 6-10 branches (flowering)(98 days)

TABLE 3 Description of Vegetative and Flowering Characteristics. PloidyNumber of sets of chromosomes Chromosome Number Number of chromosomesPlant Height Measure length of the plant from (Vegetative) top of soilmedia to highest plant growth Plant Width Plant Width was defined as how(Vegetative) wide the plant was from an aerial view. The area wasmeasured by taking 2 squares and creating a frame at the widest area ofthe plant. The measurements were recorded from where the framesoverlapped. Plant Height Measure length of the plant from (Flowering)top of soil media to highest plant growth Plant Width Class Structure ofthe overall plant Plant Height Class Height comparable with othermarigold varieties Growth Form Plant phenotype Leaf Type Type of leafLeaf Shape Structure of leaf Leaf Margin Structure of leaf edge Leafwidth (cm) Width of leaf structure from fully developed leaf frommid-section of plant Leaf Length (cm) Length of leaf structure fromfully developed leaf from mid-section of plant Leaf Color Visual colorof leaf Petiole Anthocyanin Red pigmentation of leaf stalk Stem profileShape of Stem Stem Structure Pliability of stem Node length An internodemeasured at the mid- section of the main stalk of the plant Number ofnodes below Visual number of internodes first branch below first branchNumber of first order Number of branches generating from branches themain stalk Days from planting to Observed days between sowing to firstflower first open flower Length of flowering Observed days betweensowing to season end of flowering Flowering Season Designated by numberof days to flower compared to other crops Silhouette Petal StructureNumber of Flower heads Number of flowers or buds per plant per plantFlower head diameter Diameter of stage 3 flower Flower Odor Strength offlower fragrance Flower Head Color Petal color

In an embodiment, the plant KI4662 produces zeaxanthin comprisinggreater than 2 mg/g dry weight zeaxanthin and preferably greater than 3mg/g dry weight zeaxanthin and little or no (<0.05 mg/g dry weight)lutein.

The present invention is related to the development of a novel, stable,inbred line of Tagetes erecta. This line is unique and clearly distinctfrom all other existing varieties of Tagetes erecta. Line KI4662 hasfertile male flowers and is particularly suited for use as a male inbredline which is crossed with female plants to produce hybrid seed thatwill result in plants having plant tissues, particularly flower petals,that are high in zeaxanthin and little or no lutein. The mutationpresent in KI4662 is recessive. In order for the recessive mutation tofully express in hybrid combination, both the male and the female parentshould carry the mutation. Under such circumstances, the hybrid willhave one allele from each parent and the recessive mutation would appearon both the alleles in the hybrid. For the female parent to carry themutation, mutant KI4662 should be backcrossed into the female to createa female plant that carries the mutation. Molecular markers will aid inthe “surgical” transfer and introgression of the high zeaxanthinmutation from KI4662 into the female line in order to develop a femaleline with the mutation.

Various breeding schemes may be used to produce new inbred marigoldlines from KI4662. In one method, generally referred to as the pedigreemethod, KI4662 may be crossed with another different marigold plant suchas a second inbred parent marigold plant, which either itself exhibitsone or more selected desirable characteristic(s) or imparts selecteddesirable characteristic(s) to a hybrid combination. Examples ofpotentially desired characteristics include greater flower yield, higherxanthophyll content, reduced time to crop maturity, better agronomicquality, resistance and/or tolerance to insecticides, herbicides, pests,heat and drought, and disease, and uniformity in germination times,stand establishment, growth rate, maturity and flower size. If the twooriginal parent marigold plants do not provide all the desiredcharacteristics, then other sources can be included in the breedingpopulation. Elite inbred lines, that is, pure breeding, homozygousinbred lines, can also be used as starting materials for breeding orsource populations from which to develop inbred lines.

Thereafter, resulting seed is harvested and resulting superior progenyplants are selected and selfed or sib-mated in succeeding generations,such as for about 5 to about 7 or more generations, until a generationis produced that no longer segregates for substantially all factors forwhich the inbred parents differ, thereby providing a large number ofdistinct, pure-breeding inbred lines.

In another embodiment for generating new inbred marigold plants,generally referred to as backcrossing, one or more desired traits may beintroduced into the female inbred parent marigold plant (the recurrentparent) by crossing the KI4662 plants as a male non-recurrent donorparent with another marigold plant (referred to as the recipient or-recurrent parent) which carries the gene(s) encoding the particulartrait(s) of interest to produce F₁ progeny plants. Both dominant andrecessive alleles may be transferred by backcrossing. The donor plantmay also be an inbred, but in the broadest sense can be a member of anyplant variety or population cross-fertile with the recurrent parent.Next, F₁ progeny plants that have the desired trait are selected. Then,the selected progeny plants are crossed with KI4662 to produce backcrossprogeny plants. Thereafter, backcross progeny plants comprising thedesired trait and the physiological and morphological characteristics ofmarigold inbred line KI4662 are selected. This cycle is repeated forabout one to about eight cycles, preferably for about 3 or more times insuccession to produce selected higher backcross progeny plants thatcomprise the desired trait and all of the desired physiological andmorphological characteristics of marigold inbred line KI4662 listed inTable 2 as determined at the 5% significance level when grown in thesame environmental conditions. Exemplary desired trait(s) include insectresistance, herbicide resistance, yield stability, yield enhancement andresistance to bacterial, fungal and viral disease. One of ordinary skillin the art of plant breeding would appreciate that a breeder usesvarious methods to help determine which plants should be selected fromthe segregating populations and ultimately which inbred lines will beused to develop hybrids for commercialization. In addition to theknowledge of the germplasm and other skills the breeder uses, a part ofthe selection process is dependent on experimental design coupled withthe use of statistical analysis. Experimental design and statisticalanalysis are used to help determine which plants, which family ofplants, and finally which inbred lines and hybrid combinations aresignificantly better or different for one or more traits of interest.Experimental design methods are used to assess error so that differencesbetween two inbred lines or two hybrid lines can be more accuratelydetermined. Statistical analysis includes the calculation of meanvalues, determination of the statistical significance of the sources ofvariation, and the calculation of the appropriate variance components.Either a five or a one percent significance level is customarily used todetermine whether a difference that occurs for a given trait is real ordue to the environment or experimental error. One of ordinary skill inthe art of plant breeding would know how to evaluate the traits of twoplant varieties to determine if there is no significant differencebetween the two traits expressed by those varieties. Mean trait valuesmay be used to determine whether trait differences are significant, andpreferably the traits are measured on plants grown under the sameenvironmental conditions.

Of course, the other marigold plant may be the recurrent parent wherebythe mutation associated with high zeaxanthin values and little or nolutein in KI4662 is transferred to the recurrent parent following theforegoing process. It is expected that use of such an inbred line in across with KI4662 would result in hybrid marigold plants with greaterthan 3 mg/g zeaxanthin and little or no lutein on a dry matter basis.

This method results in the generation of inbred marigold plants withsubstantially all of the desired morphological and physiologicalcharacteristics of the recurrent parent and the particular transferredtrait(s) of interest. Because such inbred marigold plants areheterozygous for loci controlling the transferred trait(s) of interest,the last backcross generation would subsequently be selfed to providepure breeding progeny for the transferred trait(s).

It should be appreciated by those having ordinary skill in the art thatbackcrossing can be combined with pedigree breeding as where inbredKI4662 is crossed with another marigold plant, the resultant progeny arecrossed back to inbred KI4662 and thereafter, the resulting progeny ofthis single backcross are subsequently inbred to develop new inbredlines. This combination of backcrossing and pedigree breeding is usefulas when recovery of fewer than all of the KI4662 characteristics thanwould be obtained by a conventional backcross are desired.

Once inbred lines are created, the next step is to determine if theinbreds have any value. This is accomplished by techniques of measuringthe combining ability of the new inbred plant, as well as theperformance of the line itself. Combining ability refers to a line'scontribution as a parent when crossed with other lines to form hybrids.Specific combining ability (SCA) refers to the ability of a line tocross to another specific inbred to form a hybrid. General combiningability (GCA) refers to the ability of a line to cross to a wide rangeof lines to form hybrids. The methodology of forming hybrids to evaluatean inbred line's contribution as a parent for the purpose of selectingsuperior lines is interchangeably known as experimental, top or testcrossing.

In accordance with processes of the present invention, a hybrid planthaving inbred KI4662 as a parent is crossed with itself or any differentmarigold plant such as an inbred marigold plant or a hybrid marigoldplant to develop a novel inbred line. For example, a hybrid marigoldplant having inbred marigold plant KI4662 as a parent may be inbred,i.e., crossed to itself or sib-pollinated, and the resulting progenyeach selfed for about 5 to about 7 or more generations, therebyproviding a set of distinct, pure-breeding inbred lines wherein each ofthe lines received all of its alleles from the hybrid marigold planthaving inbred marigold plant KI4662 as a parent. In other embodiments, ahybrid marigold plant having inbred marigold plant KI4662 as a parent iscrossed with a different marigold plant that may include any inbredmarigold plant that is not inbred marigold plant KI4662, any hybridmarigold plant that does not have KI4662 as a parent, another germplasmsource, a mutation inducing stock, or a trait donor plant, therebyproviding a set of distinct, pure-breeding inbred lines. The resultinginbred lines could then be crossed with other inbred or non-inbred linesand the resulting inbred progeny analyzed for beneficialcharacteristics. In this way, novel inbred lines conferring desirablecharacteristics could be identified.

In yet another aspect of the invention, processes are provided forproducing marigold seeds or plants, which processes generally comprisecrossing a first parent marigold plant with a second parent marigoldplant wherein at least one of the first parent marigold plant or thesecond parent marigold plant is inbred parent marigold plant KI4662. Insome embodiments of the present invention, the first inbred marigoldplant is KI4662 and is a female and in other embodiments the firstinbred marigold plant is KI4662 and is a male. These processes may befurther exemplified as processes for preparing hybrid marigold seed orplants, wherein a first inbred marigold plant is crossed with a secondmarigold plant of a different, distinct variety to provide a hybrid thathas, as one of its parents, the inbred marigold plant variety KI4662. Inthis case, a second inbred variety is selected which confers desirablecharacteristics when in hybrid combination with the first inbred line.In these processes, crossing will result in the production of seed. Theseed production occurs regardless whether the seed is collected.

Any time the inbred marigold plant KI4662 is crossed with another,different marigold inbred, a first generation (F₁) marigold hybrid plantis produced. As such, an F₁ hybrid marigold plant may be produced bycrossing KI4662 with any second inbred marigold plant. Therefore, any F₁hybrid marigold plant or marigold seed which is produced with KI4662 asa parent is part of the present invention.

When inbred marigold plant KI4662 is crossed with another inbred plantto yield a hybrid, the original inbred can serve as a paternal plantcontributing to some of the characteristics in the hybrids. Bothpaternally and maternally inherited characteristics may expressdifferently in the hybrid. However, often one of the parental plants ispreferred as the maternal plant because of its male sterility. It isgenerally preferable to use KI4662 as the male parent.

For a decision to be made to advance a hybrid, it is not necessary thatthe hybrid be better than all other hybrids. Rather, significantimprovements must be shown in at least some traits that would createvalue for some applications or markets. Some testcross hybrids areeliminated despite being similarly competitive relative to the currentcommercial hybrids because of the cost to bring a new hybrid to marketrequires a new product to be a significant improvement over the existingproduct offering.

All plants produced using inbred marigold plant KI4662 as a parent arewithin the scope of this invention, including plants derived from inbredmarigold plant KI4662. This includes plants essentially derived frominbred KI4662 with the term “essentially derived variety” having themeaning ascribed to such term in 7 U.S.C. §2104(a)(3) of the PlantVariety Protection Act, which definition is hereby incorporated byreference. This also includes progeny plant and parts thereof with atleast one ancestor that is inbred marigold plant KI4662 and morespecifically where the pedigree of this progeny includes 1, 2, 3, 4,and/or 5 or cross pollinations to inbred marigold plant KI4662, or aplant that has KI4662 as a progenitor. All breeders of ordinary skill inthe art maintain pedigree records of their breeding programs. Thesepedigree records contain a detailed description of the breeding process,including a listing of all parental lines used in the breeding processand information on how such line was used. Thus, a breeder would know ifKI4662 were used in the development of a progeny line, and would alsoknow how many breeding crosses to a line other than KI4662 were made inthe development of any progeny line. A progeny line so developed maythen be used in crosses with other, different, marigold inbreds toproduce first generation F1 marigold hybrid seeds and plants withsuperior characteristics.

Accordingly, another aspect of the present invention is methods forproducing an inbred marigold line KI4662-derived marigold plant. Thismethod for producing a KI4662-derived marigold plant, comprises: (a)crossing inbred marigold plant KI4662 with a second marigold plant toyield progeny marigold seed; and, (b) growing the progeny marigold seed,(under plant growth conditions), to yield the KI4662-derived marigoldplant. Such methods may further comprise the steps of: (c) crossing theKI4662-derived marigold plant with itself or another marigold plant toyield additional KI4662-derived progeny marigold seed; (b) growing theprogeny marigold seed of step (d) (under plant growing conditions), toyield additional KI4662-derived marigold plants; and (e) repeating thecrossing and growing steps of (c) and (d) from 0 to 7 times to generatefurther KI4662-derived marigold plants. Still further, this may compriseutilizing methods of haploid breeding and plant tissue culture methodsto derive progeny of the KI4662-derived marigold plant.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and to expressforeign genes, or additional, or modified versions of native orendogenous genes (perhaps driven by different promoters) to alter thetraits of a plant in a specific manner. Such foreign, additional and/ormodified genes are referred to herein collectively as “transgenes.” Thepresent invention, in particular embodiments, also relates totransformed versions of the claimed inbred marigold line KI4662containing one or more transgenes, particularly genes that encoderesistance to a herbicide.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

The foregoing methods for transformation would typically be used forproducing transgenic inbred lines. Transgenic inbred lines could then becrossed, with another (non-transformed or transformed) inbred line, inorder to produce a transgenic hybrid marigold plant. Alternatively, agenetic trait which has been engineered into a particular marigold lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-elite lineinto an elite line, or from a hybrid marigold plant containing a foreigngene in its genome into a line or lines which do not contain that gene.

In addition to phenotypic observations, a plant can also be described byits genotype. The genotype of a plant can be described through a geneticmarker profile which can identify plants of the same variety, a relatedvariety or be used to determine or to validate a pedigree. Geneticmarker profiles can be obtained by techniques such as RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), SimpleSequence Repeats (SSRs) which are also referred to as Microsatellites,and Single Nucleotide Polymorphisms (SNPs), Isozyme Electrophoresis andIsolelectric Focusing.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herewithin, but are envisioned to include any type ofgenetically stable marker and marker profile which provides a means ofdistinguishing varieties. In addition to being used for identificationof inbred parents, a hybrid produced through the use of KI4662 or itsparents, and identification or verification of the pedigree of progenyplants produced through the use of KI4662, the genetic marker profile isalso useful in breeding and developing backcross conversions.

DEPOSIT INFORMATION

Applicants have made a deposit of at least 2,500 seeds of inbred Tageteserecta plant KI4662 with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110 USA, under ATCCAccession No. PTA-121906. The seeds received by the ATCC on Jan. 14,2015, were taken from a deposit maintained by Kemin Industries, Inc.since prior to the filing date of this application. Access to thisdeposit will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant will make the deposit availableto the public pursuant to 37 C.F.R. §1.808. This deposit of the Marigoldinbred line KI4662 will be maintained in the ATCC depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce.

The foregoing description and drawings comprise illustrative embodimentsof the present inventions. The foregoing embodiments and the methodsdescribed herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claims are so limited. Thoseskilled in the art that have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention.

We claim:
 1. A seed of marigold inbred line designated KI4662, or a partof the seed, representative seed of the line having been deposited underATCC Accession No. PTA-121906.
 2. A substantially homogenous populationof seeds comprising at least one of the marigold seed of claim
 1. 3. Amethod for producing marigold seed, said method comprising the steps of:(a) planting the seed of claim 1 in proximity to another seed of inbredline KI4662 or to a seed of a different line; (b) growing plants fromthe seed; (c) pollinating said plants; and, (d) harvesting the resultantseed.
 4. A marigold seed produced by the method of claim
 3. 5. Amarigold plant produced by growing the seed of claim
 1. 6. A part of themarigold plant of claim 5, selected from the group consisting of anintact plant cell, a plant protoplast, an embryo, pollen, an ovule, aflower, a seed, a petal and a leaf.
 7. Pollen of the plant of claim 5.8. An ovule of the plant of claim
 5. 9. A marigold plant, or a part ofthe marigold plant, having all the physiological and morphologicalcharacteristics of the marigold plant, or a part of the marigold plant,of claim
 5. 10. A substantially homogenous population comprising atleast one of the marigold plant of claim
 5. 11. A method for producing amarigold plant, said method comprising the step of: (a) crossing inbredmarigold plant KI4662, representative seed of the line having beendeposited under ATCC Accession No. PTA-121906, with another differentmarigold plant to yield progeny marigold seed.
 12. The method of claim11, wherein the other, different marigold plant is an inbred marigoldplant.
 13. The method of claim 11, further comprising the steps of: (b)growing the progeny marigold seed from step (a); (c) underself-pollinating or sib-pollinating conditions for 5 to 7 generations;and (d) harvesting resultant seed.
 14. The method of claim 11, furthercomprising the step of selecting plants obtained from growing at leastone generation of the progeny marigold seed for a desirable trait.
 15. Amethod of introducing a desired trait from marigold inbred line KI4662,representative seed of the line having been deposited under ATCCAccession No. PTA-121906, said method comprising the steps of: (a)crossing KI4662 plants with plants of another marigold line thatcomprise a desired trait to produce F1 progeny plants; (b) selecting F1progeny plants that have the desired trait; (c) crossing selectedprogeny plants with said other marigold line plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that comprisethe desired trait and physiological and morphological characteristics ofmarigold inbred line KI4662; and (e) performing steps (c) and (d) one ormore times in succession to produce the selected or higher backcrossprogeny plants that comprise the desired trait and all of thephysiological and morphological characteristics of said other marigoldinbred line as determined at the 5% significance level when grown in thesame environmental conditions.
 16. A method for producing a hybridmarigold seed comprising crossing a first inbred parent marigold plantwith a second inbred parent marigold plant and harvesting resultanthybrid marigold seed, wherein the first inbred marigold plant or thesecond inbred marigold plant is the marigold plant of claim
 5. 17. Amethod for producing a hybrid marigold seed, the method comprising: (a)planting in pollinating proximity a seed of the inbred marigold plant ofclaim 5 and a seed of a second, different inbred marigold plant; (b)cultivating the seeds into plants that bear flowers; (c) crosspollinating at least some of the cultivated plants; and (d) harvestingseeds produced from at least one of the first or second inbred marigoldplants.
 18. The method according to claim 17, wherein the KI4662marigold plant is the male parent.
 19. The method according to claim 17,wherein the KI4662 marigold plant is the female parent.
 20. A hybridmarigold seed produced by the method according to claim
 17. 21. A hybridmarigold plant, or parts thereof, produced by growing the hybridmarigold seed of claim 20.